The actin cytoskeleton controls membrane movement during cell motility, endocytosis, and intracellular trafficking. Actin filament nucleation is known to occur at the plasma membrane and on the surface of intracellular organelles, but the spatiotemporal control mechanisms for this process are poorly understood. We have developed a cell-free model system that reconstitutes actin nucleation on the surface of endosomal vesicles. This system recapitulates a cellular signaling pathway that requires the activation and recruitment of the small GTPase, Cdc42. A close relative of the Wiskott-Aldrich Syndrome Protein, N-WASP, and the Arp2/3 complex are also recruited to the membrane, resulting in the assembly of a network of actin filaments that drives vesicle motility. The goal of this proposal is to elucidate the biochemical mechanisms underlying this process. We address the following questions: (1) What is the mechanism by which N-WASP is recruited to and activated at a membrane surface? (2) How does a symmetrical membrane surface assemble a polarized actin network capable of generating force? (3) How is cytosolic, GDP-bound Cdc42 converted into its membrane-associated, GTP-bound state?